Sequential Chlorantraniliprole– Bacillus thuringiensis Application Enhances Transcriptomic Reprogramming and Disrupts Detoxification Pathways in Spodoptera frugiperda (Noctuidae: Lepidoptera)

ABSTRACT The fall armyworm, Spodoptera frugiperda , is a highly destructive and invasive pest that poses a serious threat to agricultural productivity. Chlorantraniliprole and Bacillus thuringiensis var. kurstaki ( Bt ) are widely used for its management; however, the transcriptional responses associated with their individual and sequential applications remain insufficiently characterized. In the present study, LC 50 ‐based bioassays were conducted against third‐instar S. frugiperda larvae using a leaf‐dip method, followed by exploratory transcriptomic profiling under four treatment conditions: untreated control (Sf_C), chlorantraniliprole (Sf_Chl), Bt (Sf_Bt), and sequential chlorantraniliprole→ Bt exposure (Sf_Chl + Bt ). Chlorantraniliprole and Bt exhibited substantial larvicidal activity, with LC 50 values of 18.63 ppm and 1.30 mL L −1 , respectively. High‐quality RNA samples (RIN > 9.0) and sequencing datasets (Q30 > 90%) were obtained for all treatment groups. Exploratory transcriptomic analysis identified approximately 7500 treatment‐associated differentially expressed genes across six pairwise contrasts, with the largest number observed in the Sf_C versus Sf_Chl + Bt comparison (1985 genes; 1037 upregulated and 948 downregulated). Functional categorization revealed treatment‐associated changes in cytochrome P450 monooxygenases, glutathione S‐transferases, ATP‐binding cassette transporters, cuticle‐associated proteins, and orphan genes. Chlorantraniliprole was particularly associated with increased transcript abundance of selected CYP6‐family genes and an ABC transporter, whereas sequential exposure was associated with increased transcript abundance of selected GSTs and broader representation of metabolic and stress‐related pathways. KEGG pathway analysis further indicated treatment‐associated modulation of detoxification, energy metabolism, cellular stress, and signaling pathways, with the sequential treatment showing the broadest pathway representation. Because one RNA‐seq library was analyzed per treatment without biological RNA‐seq replication, these transcriptomic findings are considered exploratory and hypothesis‐generating rather than statistically replicated evidence of differential expression or treatment synergism. Overall, the study provides an initial molecular framework for understanding the transcriptional responses of S. frugiperda to chlorantraniliprole, Bt , and their sequential application and identifies candidate detoxification genes and pathways for validation in independently replicated experiments.